EpiMer and EpiMatrix are algorithms used to prospectively identify regions of proteins expected to bind to MHC and to be presented to T cells (cytotoxic T cells/CTL and T helper cells/Th). The algorithms have successfully identified novel clade B, C, and E epitopes from primary HIV-1 sequences. These proposed studies will elucidate cross-clade responses to putative HIV-1 T cell epitopes, evaluate the expression and recognition of the epitopes as "cassettes" in DNA plasmids, and explore the utility of including the epitopes in an HIV vaccine. The specific aims of the proposed work are to 1) Identify novel cross-reactive (cross-clade) HIV-1 CTL and Th epitopes using EpiMatrix and 2) Develop plasmids containing these epitopes as inserts, evaluate their expression and recognition in vitro. Regions of HIV that are conserved across HIV strains listed in the Los Alamos HIV Sequence Database have been identified and screened for MHC binding potential using EpiMatrix. An initial set of 25 of the highest scoring peptides that are well conserved across clades has been constructed for each of 32 class 1 alleles; funding to evaluate these putative CTL epitopes in vitro is sought. We also propose to construct a similar list of conserved, high-scoring putative class II-restricted epitopes and to evaluate these epitopes in vitro. Class I-restricted peptides that (1) bind in vitro or (2) receive high estimates of binding probability based on EpiMatrix will be selected for CTL assays to be performed at the TB/HIV Research Laboratory, using T cells derived from HIV-1 (clade B) infected individuals. Class II- restricted peptides will be studied directly in T cell assays (no other surrogate for "epitopicity" exists). Additional T cell assays will be performed by collaborators. T cell epitopes identified in this manner will be expressed as oligonucleotides and inserted in plasmids developed by Dr. Robert Whalen. In year two of the project, assays will be performed to confirm the expression and immunogenicity of the epitopes in the plasmids. These well-conserved HIV-1 CTL and Th epitopes may be useful components of a novel vector-based or plasmid-based DNA vaccine against HIV-1.